U.S. patent application number 12/238288 was filed with the patent office on 2009-07-02 for identification of thymically derived cd4 t cells by protein tyrosine kinase 7 expression.
Invention is credited to Thierry Giffon, Christopher Haines, David B. Lewis, Xiaowei Lu, Douglas T. Ross, Marc Tessier-Lavigne.
Application Number | 20090170101 12/238288 |
Document ID | / |
Family ID | 40512069 |
Filed Date | 2009-07-02 |
United States Patent
Application |
20090170101 |
Kind Code |
A1 |
Lewis; David B. ; et
al. |
July 2, 2009 |
IDENTIFICATION OF THYMICALLY DERIVED CD4 T CELLS BY PROTEIN
TYROSINE KINASE 7 EXPRESSION
Abstract
The invention provides methods of identifying naive T cells by
expression of PTK7.
Inventors: |
Lewis; David B.; (Stanford,
CA) ; Haines; Christopher; (Menlo Park, CA) ;
Giffon; Thierry; (Mountain View, CA) ; Lu;
Xiaowei; (Charlottesville, VA) ; Tessier-Lavigne;
Marc; (South San Francisco, CA) ; Ross; Douglas
T.; (Huntsville, AL) |
Correspondence
Address: |
Stanford University Office of Technology Licensing;Bozicevic, Field &
Francis LLP
1900 University Avenue, Suite 200
East Palo Alto
CA
94303
US
|
Family ID: |
40512069 |
Appl. No.: |
12/238288 |
Filed: |
September 25, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60975101 |
Sep 25, 2007 |
|
|
|
Current U.S.
Class: |
435/6.18 ;
435/29; 435/372.3; 435/6.1; 435/7.24 |
Current CPC
Class: |
G01N 2333/91205
20130101; G01N 33/56972 20130101; G01N 2333/70514 20130101; G01N
33/573 20130101 |
Class at
Publication: |
435/6 ; 435/29;
435/7.24; 435/372.3 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12Q 1/02 20060101 C12Q001/02; G01N 33/53 20060101
G01N033/53; C12N 5/08 20060101 C12N005/08 |
Goverment Interests
GOVERNMENT RIGHTS
[0001] This invention was made with Government support under
contract NIH R21 HD-37589 awarded by the National Institute of
Health. The Government has certain rights in this invention.
Claims
1. A method of identifying a mammalian naive T-cell, said method
comprising determining the level of expression of PTK7 on a
T-cell.
2. The method of claim 1, wherein the T cell is in a peripheral
blood or lymphoid tissue sample.
3. The method of claim 1, wherein the PTK7.sup.+ cell is identified
by contacting the T cell with fluorescently-labeled monoclonal
antibodies which specifically bind to PTK7 and detecting the
fluorescence of the labeled antibodies bound to the cell.
4. The method of claim 1, wherein the T cell is a CD4.sup.+
cell.
5. The method of claim 1, wherein the T cell is a recent thymic
emigrant.
6. A method of making an isolated population of naive T cells, said
method comprising obtaining a T-cell sample; determining the level
of expression of PTK7 on the surface of the T cell; and isolating
the PTK7.sup.+ T cells from those T cells that are PTK7.sup.-.
7. The method of claim 6, wherein the biological sample is
contacted with a labeled monoclonal PTK7 antibody, and the cells
are sorted according to the amount of the labeled antibody bound to
them, wherein cells having bound antibody are identified as
PTK7.sup.+ cells and are isolated from cells having lacking
antibody bound to them.
8. A method of analyzing a patient sample for the presence of naive
T cells, the method comprising: quantitating the presence of PTK7
protein or mRNA in a test sample, wherein the presence of PTK7 as
compared to a control sample lacking naive T cells is indicative of
the presence of naive T cells in the test sample.
9. The method of claim 8, wherein the sample selected from
peripheral blood, lymph node, spleen and cord blood.
10. The method of claim 9, wherein the sample is dried blood.
11. The method of claim 10, wherein the sample is obtained from an
individual suspected of having an immunodeficiency.
12. The method of claim 8, wherein the test sample is peripheral
blood dried on paper.
13. The method of claim 12, wherein the patient is undergoing
immunosuppressive treatment.
14. The method of claim 12, wherein the patient is undergoing
immune restoration treatment.
15. The method of claim 12 wherein the patient is suspected of an
immunodeficiency.
16. An isolated population of PTK7+ naive T cells.
17. The isolated cell population of claim 16, wherein the cells are
CD4+.
18. The method of claim 16, wherein the cells are recent thymic
emigrants.
Description
BACKGROUND OF THE INVENTION
[0002] T lymphocytes are cells of the adaptive immune system that
mainly develop in the thymus and are selected to express a unique
and exquisitely-specific T-cell receptor (TCR) that will both
remain tolerant (non-responsive) to self antigens, and yet bet able
to recognize and respond to a foreign linear peptide in the context
of self-major histocompatibility complex (MHC) molecules.
[0003] During differentiation, progenitors to T cells arise from
stem cells in the bone marrow, and are then recruited to the
thymus, entering via vessels in the perimedullary cortex, where
they begin differentiation into T cells. Following a series of
ligand/receptor and cytokine or chemokine/receptor interactions,
the initial "double negative" thymocytes progress through
maturation, eventually rearranging T-cell receptor genes to
generate a productive TCR.beta. chain gene. The TCR.beta. chain
protein is coexpressed with the pre-T alpha receptor, and this
triggers co-expression of both the CD4 and CD8 coreceptors by cells
that are referred to as double positive thymocytes (DPs). Upon a
productive TCR.alpha. rearrangement, the TCR.beta. and TCR.alpha.
chain proteins are coexpressed as a heterodimer in association with
proteins of the CD3 complex. Positive selection then occurs,
resulting in the upregulation of the surface levels of the
.alpha..beta. TCR/CD3 complex and loss of either CD8 to form
CD4+CD8- thymocytes (MHC class II selection), or loss of CD8 to
form CD4-CD8+ thymocytes (MHC class I selection). These
single-positive thymocytes move into the medulla where negative
selection occurs to purge the repertoire of autoreactive T
cells.
[0004] Following selection, these naive T cells are exported from
the thymus. The rate of thymic export appears to remain constant
throughout the lifetime of the individual but the volume of export
is proportional to the volume of residual thymic mass index.
Therefore, as individuals age, their thymus involutes and is
largely converted into fat, and there is a concomitant decrease in
the number of recent thymic emigrants that are exported on a daily
basis. Despite this reduction in thymic output throughout the time
course of life in individuals, the absolute number of T cells in
the peripheral T-cell pool remains surprisingly constant throughout
life.
[0005] Following thymic development, CD4.sup.+ and CD8.sup.+ T
cells are exported into the peripheral circulation as naive T
cells, which survey self-major histocompatibility complex (MHC)
molecules for foreign peptide antigens. In response to cognate
antigen, they differentiate into effector and memory T lymphocytes.
The developmental stage of human T-cell maturation following export
from the thymus has not been extensively characterized. A limited
number of studies in other species have suggested that recent
thymic emigrants (RTEs) are not fully mature and require a
developmental period of post-thymic maturation to become fully
functional T cells. In the rat, lack of both RT6 and CD45RC
expression distinguish RTEs from the rest of the mature T cell
pool. Unfortunately, human homologs for markers such as RT6 or
CD45RC have not been identified for humans, and experimental
approaches useful in animals, such as ectopic GFP expression or
intrathymic injections of dyes, are not possible. Therefore,
aspects of human post-thymic T cell development remain unknown.
[0006] In order to overcome these challenges, investigators have
developed a surrogate marker for RTE frequency in human T cell
populations called sjTREC content analysis. sjTRECs, or signal
joint T-cell receptor excision circles, are the signal joint
byproducts of VDJ recombination leftover following TCR
rearrangement in the thymus. Because they lack an origin of
replication, they are not replicated during each cell division, and
as such are diluted in T cells following cell proliferation.
Relative or absolute TREC content can be used to determine the
relative "age" of bulk populations of cells, and to roughly
estimate the contribution of thymic output to the T-cell pool.
However, TRECs can only be detected with a PCR assay that requires
killing the cells; the TREC assay cannot determine at a single cell
level whether a T cell is an RTE; and because a TREC has to be
split between two daughter cells, even TREC.sup.+ naive T cells may
have undergone some rounds of division. TRECs are also assumed to
be equally stable in various naive T-cell populations in the
absence of cell proliferation, but whether this is actually the
case remains unknown.
[0007] A unique cell-surface marker that directly detects naive T
cells, particularly live RTEs, at the single cell level is highly
desirable for enumerating these cells and understanding their
function. The ready identification of these cells in humans and an
understanding of their maturation and functional attributes are
important not only for understanding the basic biology of T-cell
development and function, but also for the development of better
vaccines for human neonates and infants. Furthermore, such a marker
would allow one to monitor the kinetics of thymic output in normal
patients over the adult lifespan, and T-cell reconstitution in
those patients with transient deficiencies in CD4+ T cells, such as
those with HIV-infection, hematopoietic stem cell transplantation,
or chemotherapy treatment, and those with inherited
immunodeficiencies involving T cells, such as severe combined
immunodeficiency (SCID).
BRIEF SUMMARY OF THE INVENTION
[0008] Methods are provided for the detection, identification and
isolation of naive T cells. It is shown herein that thymocytes and
naive T cells, e.g. recent thymic emigrants, express on their cell
surface protein tyrosine kinase 7 (PTK7), a member of the receptor
tyrosine kinase family. The four major subpopulations of murine and
human thymocytes express PTK7 mRNA and protein, which decreases
with maturation. A discrete subset of human peripheral naive T
cells express PTK7. The detection of RTE cells in the peripheral
blood provides a sensitive method for assessment of thymic
function, and finds use in monitoring the status of individuals
undergoing chemotherapy; treatment for HIV infection; and other
chronic or acute conditions affecting the generation of naive T
cells. The detection of RTE cells in the peripheral blood also
provides a sensitive s method of monitoring the status of patients
treated to improve immune function, e.g. following hematopoietic
stem cell transplantation, cytokine immunotherapy, etc.
[0009] In some embodiments, the invention provides methods of
determining whether a T cell in a biological sample is a naive T
cell by detecting the cell surface antigen PTK7. In other
embodiments, the invention provides methods of identifying naive T
cells by identifying PTK7.sup.+ T cells according to their level of
expression of the PTK7 biomarkers in a biological sample. In some
embodiments, the T cells are CD4.sup.+ T cells. In other
embodiments, the T cells are CD8.sup.+ T cells. Such cells may be
further classified as recent thymic emigrants. Human cells are of
particular interest, as are CD4.sup.+ cells. A biological sample of
particular interest is peripheral blood.
[0010] In another embodiment, the presence of naive T cells in a
biological sample is determined by quantitating the level of PTK7
in the sample. Any convenient method may be used for the
quantitation of PTK7, including detection of the protein by
immunoassay, e.g. ELISA, immunohistochemistry, flow cytometry,
etc.; detection of PTK7 mRNA, etc.
[0011] In other embodiments, the invention provides methods of
making an isolated population of naive T-cells, which may be
CD4.sup.+ T cells, and which may further be RTE T cells. In one
embodiment, the population is isolated by obtaining a biological
sample comprising T-cells and determining the level of expression
of PTK7 on the surface of the T-cells and isolating the PTK7.sup.+
T-cells from those T-cells which are PTK7.sup.-. The cells may be
further separated on the basis of CD4 and/or the CD8
expression.
[0012] In another embodiment, the invention provides kits
comprising materials specifically useful in performing the above
methods. A kit for identifying or isolating a naive T-cell
population, for example, can comprise an antibody that binds PTK7.
The label is preferably labeled (e.g., a fluorescent, magnetic or
isotopically label). In addition, the kit may further provide
instructions for formulating the T-cell population in a suitable
media for contacting with cells in vivo or in vitro.
[0013] In another aspect, the invention provides methods for
identifying immunosuppressive, particularly thymus-damaging drugs
by determining their effect on PTK7.sup.+ cells, e.g. PTK.sup.+
cells in the peripheral blood. As PTK7+ cells are typically recent
thymic emigrants, such detection provides a sensitive means of
monitoring thymic function.
[0014] In another aspect, the invention provides methods for
assessing the immunostimulatory impact on thymic function of drugs,
such as the cytokines IL-2, IL-7, or TSLP, by determining the
levels of PTK7.sup.+ cells in the peripheral blood before and after
treatment intervention.
[0015] In another aspect, the invention provides a means to isolate
RTEs by means of antibody staining of surface PTK7, and using these
cells as a means of adoptive immunotherapy, such as in settings of
T-cell lymphopenia.
[0016] In another aspect, the invention provides a means to screen
blood samples in newborns for severe inherited T-cell
immunodeficiency, such as severe combined immunodeficiency, by
analyzing blood spots for PTK7 protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1. Human CD4+CD8- thymocytes and cord blood (neonatal)
CD4+CD45RA.sup.hi peripheral T cells (which are enriched in RTEs)
contain higher levels of TRECs and higher levels of PTK7
transcripts than adult naive CD4+CD45RA.sup.hi T cells. (A). Cells
were either fluorescent activated cell sorter (FACS)-purified or
magnetic activated cell sorter (MACS)-purified to >90% purity,
then total DNA was extracted, and a real-time PCR based assay for
detecting sjTRECs was performed, after normalization for cell
number based on real-time PCR detection of a conserved TCR alpha
genomic sequence. (B). A separate aliquot of the same populations
of cells was used for RNA extraction, then 250 ng was used to make
cDNA using reverse transcriptase and random hexamers, and the cDNA
was used in a real-time PCR based assay for detecting PTK7
transcripts. 18S rRNA serves as a normalization control between
samples for cell number. A separate cDNA production reaction with
250 ng of RNA and no reverse transcriptase added was used as a
negative control for amplification from genomic DNA, and there was
>5 Ct difference (minimal amplification) of 18S rRNA under this
condition.
[0018] FIG. 2. PTK7 is expressed on human thymocytes. (Shaded
region=unstained, dashed line=isotype-stained, solid line=anti-PTK7
stained cells.) (A). Expression is highest on the most immature
cells (CD4-CD8- thymocytes), and decreases as the cells mature
through the CD4+CD8+, and CD8+CD4- or CD8-CD4+ stages. (B). The
maturation-specific expression level of PTK7 is further shown in
the CD3 versus PTK7 dot plots. The PTK7.sup.hi expressing cells in
the bimodal pattern of the CD4+CD8- population represents the
immature single positive ("ISP") thymocytes that are CD3- and are a
transitional population between CD4-CD8- and CD4+CD8+ thymocytes.
(C). The CD1.sup.low thymocytes that are CD3.sup.hi are about to be
exported to the periphery and retain PTK7 expression above the
level in the isotype-stained control. The small percentage of
PTK7-CD1- cells may be thymic re-entrants. The figures are a
representative example of three experiments on different human
thymi.
[0019] FIG. 3. PTK7 is expressed on a small population of human
adult peripheral blood CD4+CD45RA.sup.hi naive T cells and on the
majority of neonatal cord blood CD4+ T cells. (Shaded
region=unstained, dashed line=isotype-stained, solid line=anti-PTK7
stained cells.) (A). Cells shown in histograms are gated on
lymphocytes (based on forward scatter (FSC) and side scatter (SSC))
and either CD4+CD45RA.sup.hi or CD4+CD45RA.sup.lo cells. The
percentages shown in the histograms represent the anti-PTK7
specific staining % minus the isotype-stained % s (i.e.,
16.2-6.2=10% for CD45RA.sup.hi cells, and 5.3%-4.8%=0.5% for the
CD45RA.sup.lo cells. (B) A panel of % s of PTK7.sup.+ cells from a
total of 3 umbilical cord blood samples (N=3) and 4 adult donors
(N=4). Bars represent the mean of the donors, and the bars
represent the standard deviation for the donors for the
CD45RA.sup.hi and CD45RA.sup.lo populations, respectively.
[0020] FIG. 4. PTK7+ T cells are recent thymic emigrants. (A).
Either lymphocyte CD4+CD45RA.sup.hi cells or their CD45RA.sup.lo
counterparts were gated as before, then co-analyzed for CD31 and
PTK7 surface expression by flow cytometry after staining. All PTK7+
cells are also CD31+, indicating that the CD31- population, which
has undergone homeostatic proliferation, does not contain any PTK7+
cells. Co-staining with CD5 and the an irrelevant IgG1 isotype
control for the CD5 and CD31 antibodies verifies that PTK7 stain is
uniform across a positively staining and a negatively staining
population, where expression of the second marker is uniform and
independent of the maturation state of the cell. These data are
representative of six separate experiments with four different
adult donors. (B). Cells were stained as previously described, and
sorted into three separate populations (PTK7+CD31+=I,
PTK7-CD31+=II, and PTK7-CD31-=III). Total DNA was isolated from the
sorted cells and subjected to analysis for TCR.alpha. and sjTREC
levels. Unfractionated infant human thymocytes and cord blood CD4+
T cells are the positive controls shown in the first two lanes, and
K562 represents the negative control, an erythroleukemia cell line
that contains no TRECs. Absolute #s of TREC levels were determined
by correcting each sample for cell # based on TCR.alpha. levels.
Bars represent the means of triplicates, and the error bars are
standard deviation. This experiment was repeated three times for a
single donor (age 36), and similar results were obtained for two
separate donors (aged 25 and 31).
[0021] FIG. 5. CD4+CD25+FoxP3+ regulatory T cells, most of which
have undergone homeostatic proliferation to self antigens in the
periphery, no longer express PTK7. Cells were stained for CD4,
CD25, and PTK7 as before, then fixed and permeabilized with an
intracellular staining protocol for the FoxP3 transcription factor.
The majority of the PTK7+ cells are in the CD25 gate and are FoxP3
negative, i.e., they are not regulatory T cells, although there are
some CD25+FoxP3- cells. This is a representative experiment from
two separate adult donors.
[0022] FIG. 6. PTK7+RTEs exhibit immaturities in immune function,
including clonal expansion in response to a TCR/CD3 stimulus in
vitro. (A). PTK7+ and PTK7' naive T cells were sorted and placed in
culture with anti-CD3/CD28 beads and stimulated for 72 and 96
hours. .sup.3H-thymidine incorporation was assessed in the final 16
hours of culture. PTK7+ cells did not proliferate as well as PTK7-
cells at all timepoints tested. (B). Simple binding of the
anti-PTK7 antibody to endogenous PTK7 expressed on the cell surface
does not impair the proliferative response of cord blood CD4+
purified T cells (a population that uniformly expresses relatively
high levels of PTK7) to anti-CD3/CD28.
[0023] FIG. 7. PTK7+ RTEs retain thymocyte-like properties such as
the ability to respond by proliferating to IL-7 in vitro. (A).
PTK7+ and PTK7- sorted cells were placed in culture with 10 ng/ml
recombinant hIL-7 for 5 days and assayed for .sup.3H-thymidine
incorporation in the final 16 hours of culture. (B). Simple binding
of the anti-PTK7 antibody to endogenous PTK7 expressed on the cell
surface does not alter the proliferative response of cord blood
CD4+ purified T cells (a population that uniformly expresses
relatively high levels of PTK7) to rhIL-7. (C). The lack of ability
of PTK7- cells to respond to IL-7 is not due to an inability to
express the IL-7R alpha chain.
[0024] FIG. 8. PTK7+RTEs display immaturities in the ability to
secrete IL-2 and the T helper 1 (Th1) signature cytokine
interferon-gamma (IFN-.gamma.). (A) PTK7+ and PTK7- naive T cells
were sorted and placed in culture with anti-CD3/CD28 beads and
stimulated for 12 hours, followed by an intracellular cytokine
stain. (B) Supernatants were collected and assayed by sandwich
ELISA at the indicated timepoints. For the IFN-.gamma., the sorted
cells were co-incubated with autologous CD14+ MACS-purified
monocytes and cultured for 3 days in the presence of anti-CD3/CD28
beads.
[0025] FIG. 9. PTK7 is expressed on murine thymocytes. (Light line
in the histogram=unstained cells, gray line=isotype-stained cells,
and black line=anti-PTK7 stained cells.) Expression is highest on
the most immature cells (CD4-CD8- thymocytes), and decreases as the
cells mature through the CD4+CD8+, and CD8+CD4- or CD8-CD4+ stages.
The maturation-specific expression level of PTK7 is further shown
in the plots with CD3 and CD24. The PTK7 expressing cells in the
bimodal pattern the of CD8+CD4- plot represent the population of
immature CD8+C4- single positive thymocytes which are CD24.sup.high
and CD3.sup.low and are an intermediate between CD4-CD8- and
CD4+CD8+ thymocytes in most mouse strains. These data are a
representative example of four experiments on different mouse
thymi.
[0026] FIG. 10. PTK7 polyclonal anti-serum specifically binds a
PTK7-GFP transfected cell line, and does not bind PTK7-/- murine
thymocytes. (A) PTK7 polyclonal anti-serum specifically binds a
PTK7-GFP transfected cell line. DNA sequence encompassing the
extracellular and transmembrane regions of mouse PTK7 was cloned by
RT-PCR from murine thymus RNA. It was then cloned into pEGFP-N3,
which contains the CMV immediate early promoter and creates a GFP
fusion protein, with eGFP at the C-terminus of the protein, and
hence on the cytoplasmic side of the cell (PTK7 is a Type I
transmembrane protein). CHO-P cells were then transfected by
electroporation with pEGFP-N3-PTK7. The staining experiment was
repeated once with similar results. (B) Anti-PTK7 does not bind to
PTK7-/- murine thymocytes. Embryonic day 18 (E18) murine thymocytes
from wild type (solid line), heterozygous (gray line), and
knock-out (light gray line) embryos were stained with the anti-PTK7
serum. The intermediate staining of the heterozygous thymocytes
likely indicates that PTK7 transcripts are normally generated from
both homologous chromosomes. Unstained (shaded region), and
Isotype-stained wild type thymocytes (dashed line) are also
displayed as negative controls. CD4 and CD8 dot plots of adult and
embryonic thymocytes are shown for comparison to indicate the lack
of maturity of the E18 thymocytes. This experiment is a
representative example from three separate experiments.
[0027] FIG. 11. The anti-mouse PTK7 anti-serum specifically
recognizes both mouse and human PTK7 proteins in both native (flow
cytometry) and denatured (western blot) protein states. A). Total
RNA was extracted from both mouse and human thymocytes and
peripheral T cells, and cell lines, and was reverse-transcribed
into cDNA. Quantitative real-time PCR was performed with SyberGreen
detection using primers to a C-terminal sequence of human PTK7, and
an N-terminal sequence of mouse PTK7. B). Jurkat (human) and EL-4
(murine) cell lines were stained with the anti-PTK7 antiserum
(shaded region=unstained, dashed line=isotype-stained, solid
line=anti-PTK7 stained cells). The quantitative PCR graphs
correspond directly to the flow cytometry profiles showing PTK7
protein expression specifically in Jurkat T cells and not EL-4
cells. The anti-PTK7 serum also binds denatured PTK7 protein in a
western blot format. Total cell lysates from 1) EL-4 cell line, 2)
Jurkat cell line, 3) mouse spleen, 4) recombinant PTK7, 5) mouse
brain, and 6) mouse thymus.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0028] Unless otherwise stated, the following terms used in the
specification and claims have the meanings given below.
[0029] It is noted here that as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural reference unless the context clearly dictates otherwise.
[0030] By a "population of cells" is meant a plurality of cells,
preferably at least 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6,
10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, or 10.sup.11 cells. The
population in some embodiments has from 10.sup.5 to 10.sup.7 cells,
10.sup.6 to 10.sup.8 cells, or from 10.sup.8 to 10.sup.11 cells, or
10.sup.10 to 10.sup.12 cells.
[0031] Immune conditions, diseases, disorders and reactions or
responses to be monitored or treated according to the methods and
compositions of the invention means a disease in which the immune
system contributes to pathogenesis. Of interest are immune
conditions where there is a deficiency in the generation of T
cells, particularly relating to thymic maturation of T cells.
Thymic activity is compromised by certain viral infections,
including HIV infection, by immunosuppressive drugs including
chemotherapy, by inborn errors of metabolism and other genetic
defects, thymectomy, and the like. Also of interest are treatments
to restore immune function, e.g., hematopoietic stem cell or thymus
transplantation or cytokines, such as IL-2, IL-7, and TSLP.
[0032] The response of a patient to treatment may be monitored by
determining the frequency of naive, particularly RTE, T cells in a
sample from a patient. A decrease in the number of naive, or RTE, T
cells in the sample may be used as an indication to decrease
immunosuppressive treatment. An increase in RTEs in a sample may be
used as an indication to reduce immunostimulatory treatment, such
as with cytokines.
[0033] "Recent thymic emigrant" refers to a T cell in tissues other
than the thymus, which T cells are typically antigenically naive,
that is they have not yet been stimulated by an antigen presenting
cell in an MHC+foreign peptide antigen context. A naive T cell may
be defined as a recent thymic emigrant when it has emigrated from
the thymus not more than 6 months earlier, not more than 4 months
earlier, not more than 2 months earlier, not more than 1 month
earlier, or less. In some embodiments of the invention an RTE may
have emigrated from the thymus not more than 2 months earlier.
[0034] "CD," "cluster of differentiation" or "common determinant"
as used herein refers to cell surface molecules recognized by
antibodies. Expression of some CDs (e.g., CD4, CD8) is specific for
cells of a particular lineage or maturational pathway, and the
expression of others varies according to the state of activation,
position, or differentiation of the same cells. Preferably, in some
embodiments, the CD determinants are human when the isolated cells
are to be administered to a human or a human immune response is
being studied.
[0035] As used herein, the term PTK7 refers to the "protein
tyrosine kinase 7" protein, which as shown herein is present on
thymically derived naive T cells. The PTK7 protein is described in
the literature. The gene for human protein tyrosine kinase 7
(PTK7), which is also called CCK-4 or colon carcinoma kinase-4, was
originally cloned in 1995 by Mossie's group in an effort to
identify novel proteins involved in colon tumorigenesis (see Mossie
et al. (1995) Oncogene 11: 2179-2184; and Park et al. (1996) J.
Biochem. 119: 235-239, each herein specifically incorporated by
reference). PTK7 shares similarities with other PTK family members,
but it contains several key mutations in conserved regions that
prevent it from being an active kinase, including the DFG motif,
the GxGxxG region, and the HRDL domain. Many of these residues are
crucial for ATP binding in the cytoplasmic domain so that it is
predicted to be a "dead" kinase. The protein structure contains 7
Ig-like domains in the extracellular portion, a conserved .about.20
amino acid single pass transmembrane region, and a .about.300 amino
acid cytoplasmic region that contains many N-glycosylation sites
and 6 tyrosines.
[0036] The term "PTK7.sup.+" refers to cells which stain when
treated with a labeled antibody directed toward PTK7. Generally,
cells are distinguished according to their PTK7 expression levels
based upon readily discernible differences in staining intensity as
is known to one of ordinary skill in the art. In some embodiments,
the cut off for designating a cell as a PTK7.sup.+- cell can be set
in terms of the fluorescent intensity distribution observed for the
cells as compared to an isotype-matched control. A PTK7.sup.- cell
can be designated as one which does not stain any more brightly
than the staining with an isotype matched control. The designation
of a cell type with respect to its levels of expression of a
recited biomarker or CD is meant to describe the cell being
referenced by its biomarker expression phenotype and is not
necessarily an indicator that expression levels were actually
determined for the referenced cell.
[0037] PTK7.sup.+ naive T-cell populations for use according to the
invention are cell populations which have been positively selected
for the PTK7 biomarker. In some embodiments, the cells have been
further characterized with respect to other CD determinants,
particularly the CD4 determinants (e.g., positively or negatively
selected for with respect to CD4). In other embodiments, the cells
have been further characterized according to their expression of
common determinants other than CD4, including CD3, and CD8, where
the PTK7 cells may be CD3.sup.+ T cells, or CD8.sup.+ T cells. In
preferred embodiments, the cell populations are substantially the
selected cell type.
[0038] As used herein, the term "CD4" refers to a cell-surface
glycoprotein typically found on the mature helper T cells and
immature thymocytes, as well as on monocytes and macrophages. On T
cells, CD4 is the co-receptor for the T cell receptor (TCR) and
recruits the tyrosine kinase Ick. With its D1-portion, CD4 can
attach to the .beta.2-domain of MHC class II molecules. CD4.sup.+
refers to cells which stain brightly when contacted with labeled
anti-CD4 antibody, and CD4.sup.- refers to cells of a type which
stain the least brightly, dull or not at all, when contacted with a
fluorescently labeled CD4 antibody. Generally, the cells are
distinguished according to their CD4 expression levels based upon a
readily discernible differences in staining intensity as the CD4
staining is clearly bimodal. In some embodiments, the frequency
distribution of the CD4 staining is obtained for all the cells and
the population curve fit to a higher staining and lower staining
population, and cells assigned to the population to which they most
statistically are likely to belong in view of a statistical
analysis of the respective population distributions. In some
embodiments, the CD4.sup.- cells stain two to three fold less
intensely than the CD4.sup.+ cells. Particularly preferred methods
are also exemplified in the Examples.
[0039] Methods of segregating CD4 T cells into + and - categories
are known to persons of ordinary skill in the art. In some
embodiments, the frequency distribution of the CD4 staining is
obtained for all the cells and the population curve fit to a higher
staining and lower staining population, and cells assigned to the
population to which they most statistically are likely to belong in
view of a statistical analysis of the respective population
distributions. In some embodiments, the CD4.sup.+ cells stain two-
to three-fold more intensely than the CD4.sup.- cells.
[0040] As used herein, the term "sample" or "biological sample"
refers to tissues or body fluids removed from a mammal, preferably
human, and which contain naive T cells, including, but not limited
to, PTK7.sup.+ T-cells. In some embodiments, the samples are taken
from individuals with an immune response which needs to be
monitored. In some embodiments, the individual has an
immunodeficiency. In some embodiments, the individual is being
treated with an agent that is immunosuppressive, e.g. a
chemotherapeutic agent. In other embodiments the individual is
being treated with an agent that restores immune function, e.g.
anti-viral therapy such as HAART; stem or progenitor cell
replacement, thymus transplant, or immunostimulatory drugs, e.g.,
cytokines. Samples preferably are blood and blood fractions,
including peripheral blood. The biological sample is drawn from the
body of a mammal, such as a human, and may be blood, cord blood, or
similar tissues or cells. Methods for obtaining such samples are
well known to workers in the fields of cellular immunology and
surgery. They include sampling blood in well known ways, or
obtaining biopsies from the thymus or other tissue or organ.
[0041] The term "isolated" with regard to a population of cells as
used herein refers to a cell population which either has no
naturally-occurring counterpart or has been separated or purified
from other components, including other cell types, which naturally
accompany it, e.g., in normal or diseased tissues such as lung,
kidney, or placenta, tumor tissue such as colon cancer tissue, or
body fluids such as blood, serum, or urine. Typically, an isolated
cell population is at least two-fold, four-fold, or eight-fold
enriched for a specified cell type when compared to the natural
source from which the population was obtained.
[0042] A population or subpopulation of cells which is
"substantially" of a specified cell type is one which has a count
of the specified cell type which is at least 50%, 75%, 80%, 90%,
95% or, most preferably, 98% or 99% of the total cell count of the
population or subpopulation or one which is at least two-fold,
four-fold, eight-fold, ten-fold or 20-fold enriched for a specified
cell type as compared to a source population of the specified cell
type. A substantially pure population may be at least about 50%
PTK7.sup.+ T cells in the population, at least about 75% PTK7.sup.+
T cells in the population, at least about 80% PTK7.sup.+ T cells in
the population, at least about 90% PTK7.sup.+ T cells in the
population, or more. The term T cells as used herein refers to
cells having a functional rearranged T cell receptor, which cells
may be characterized as expressing CD3.
[0043] An "anti-X antibody" or "X antibody" according to the
invention is an antibody which can specifically bind to X. For
instance, the anti-PTK7 antibody or PTK7 antibody is capable of
binding PTK7. The antibodies for use according to the invention
include, but are not limited to, recombinant antibodies, polyclonal
antibodies, monoclonal antibodies, chimeric antibodies, human
monoclonal antibodies, humanized or primatized monoclonal
antibodies, and antibody fragments. A great many lymphocyte
biomarker specific antibodies are commercially available.
[0044] "Antibody" refers to a polypeptide comprising a framework
region from an immunoglobulin gene or fragments thereof that
specifically binds and recognizes an antigen. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon, and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
Typically, the antigen-binding region of an antibody will be most
critical in specificity and affinity of binding.
[0045] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
[0046] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well-characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially Fab with part of the
hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993).
While various antibody fragments are defined in terms of the
digestion of an intact antibody, one of skill will appreciate that
such fragments may be synthesized de novo either chemically or by
using recombinant DNA methodology. Thus, the term antibody, as used
herein, also includes antibody fragments either produced by the
modification of whole antibodies, or those synthesized de novo
using recombinant DNA methodologies (e.g., single chain Fv) or
those identified using phage display libraries (see, e.g.,
McCafferty et al., Nature 348:552-554 (1990))
[0047] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein,
often in a heterogeneous population of proteins and other
biologics. Specific binding to an antibody under such conditions
requires an antibody that is selected for its specificity for a
particular protein. For example, polyclonal antibodies can be
selected to obtain only those polyclonal antibodies that are
specifically immunoreactive with the selected antigen and not with
other proteins. This selection may be achieved by subtracting out
antibodies that cross-react with other molecules.
[0048] Preferably a "label" or a "detectable moiety" is covalently
or noncovalently attached to the antibody. A label may be
detectable by spectroscopic, photochemical, biochemical,
immunochemical, chemical, or other physical means. Particularly
useful labels are fluorescent dyes. Methods of attaching labels to
antibodies are well known to those of ordinary skill in the art.
Particularly preferred labels are those which are attached to the
antibody by a linker which can be readily cleaved or separated or
subject to hydrolysis by contact with a predetermined enzyme under
physiological conditions. The antibody may also be conjugated with
a magnetic particle, such as a paramagnetic microbead (Miltenyi
Biotec, Germany). An activated T cell bound by a magnetically
labeled antibody may be isolated using techniques including, but
not limited to, magnetic cell sorting. Suitably labeled antibodies
to PTK7, CD4, CD8, CD3, as well as many other CDs, are commercially
available and known to one of ordinary skill in the art. The
antibody may be labeled before or after contact with the sample or
before or after contact with the CD. The CD antibody may be labeled
by contacting with a labeled antibody which binds to the
CD-antibody.
[0049] Immunologic detection. As used herein the term refers to
detecting, particularly to quantitating, the number of protein
molecules of interest in a sample, or of cells comprising a protein
of interest in a sample, by specific binding of the protein to an
antibody or fragment thereof. Methods of immunologic detection are
known in the art, and include, for example, "bulk" assays that
utilize a cell lysate, e.g. RIA, ELISA, and various sandwich assay
formats. Other methods of immunologic detection are cell specific,
e.g. immunohistochemistry, cell staining and FACS analysis, and the
like.
[0050] As an alternative to immunologic detection, certain methods
of the invention may utilize nucleic acid detection, particularly
detection of specific mRNA, in any convenient hybridization format.
Such methods may be used to quantitate the presence of PTK7
specific expression in a sample at the mRNA level.
Embodiments
[0051] Methods are provided for the detection, identification and
isolation of naive T cells by determining the expression of PTK7.
PTK7.sup.+ T cells are shown herein to be naive T cells, and are
found in peripheral blood samples from pre-natal, neonate, to adult
samples. Expression is particularly high in young individuals, e.g.
neonates, but can be clearly detected at all ages. The PTK7+ naive
T cells are usually recent thymic emigrants, particularly in
post-natal individuals, e.g. children older than neonates, adults,
elderly, etc. The naive T cells also express various markers for T
cells, including a rearranged T cell antigen receptor, e.g. an
.alpha./.beta. T cell antigen receptor; CD3, CD4 or CD8, etc., and
in some embodiments the sample is also analyzed for expression of a
T cell marker. The analysis will include suitable controls against
which the expression of a test sample is compared, e.g. positive
controls, negative controls, including isotype matched antibody
staining for immunologic detection, and the like as known in the
art.
[0052] It is a feature of the invention that naive T cells can be
identified in peripheral blood samples with a single marker, i.e.
with expression of PTK7. Samples may be gated by size and scatter
for lymphocytes.
[0053] For many embodiments, PTK7 is detected in peripheral blood
samples, although for some purposes thymocytes samples, or other
sources of lymphocytes may be used. The sample is obtained from any
mammalian species, e.g. equine, bovine, porcine, canine, feline,
rodent, e.g. mice, rats, hamster, primate, etc., particularly
human. The sample may be obtained from a live donor, or freshly
frozen tissue, a dried blood sample, tissue frozen within about 12
hours of death and maintained at below about -20.degree. C.,
usually at about liquid nitrogen temperature (-180.degree. C.)
indefinitely, or maybe a lysed cell sample, e.g. a Guthrie card
blood sample from a neonate.
[0054] In some embodiments the individual from which the sample is
obtained has or is suspected of having an immune dysfunction,
particularly an immune dysfunction affecting thymic function, for
which monitoring of naive T cell generation is of interest. Such
individuals include those being screened for immune dysfunction,
e.g. neonatal screening for immunodeficiency; for early detection
of immune dysfunction in HIV+ individuals; for early detection of
thymic damage related to chemotherapy, and the like. Such
individuals also include those having a known immune dysfunction,
where monitoring of thymic activity is of interest, e.g. during
stem cell reconstitution, during treatment with cytotoxic and/or
immunosuppressive agents, during treatment of HIV infection, and
the like. In other aspects the invention provides analysis of
individuals receiving immune stimulatory therapy.
[0055] Where the analysis is based on a dried or lysate sample,
e.g. a cord blood lysate, biopsy sample lysate, peripheral blood
lysate, etc. detection of PTK7 expression may utilize various means
of determining the expression of PTK7, including immunologic and
nucleic acid detection methods. In some embodiments, the presence
of mRNA encoding PTK7 in a sample is detected.
[0056] Any suitable qualitative or quantitative methods known in
the art for detecting specific mRNAs can be used. mRNA can be
detected by, for example, hybridization to a blot or array, in situ
hybridization in tissue sections, by reverse transcriptase-PCR, or
in Northern blots containing poly A.sup.+ mRNA. One of skill in the
art can readily use these methods to determine the presence of a
specific mRNA transcript in a sample. Methods of detecting specific
transcripts include serial analysis of gene expression (SAGE)
methodology (Velculescu et al., Science (1995) 270:484);
differential display (DD) methodology as set forth, for example in
U.S. Pat. No. 5,776,683; and U.S. Pat. No. 5,807,680; and
hybridization analysis.
[0057] Hybridization analysis is based on the specificity of
nucleotide interactions. Oligonucleotides or cDNA can be used to
selectively identify or capture DNA or RNA of specific sequence
composition, and the amount of RNA or cDNA hybridized to a known
capture sequence determined qualitatively or quantitatively, to
provide information about the relative representation of a
particular message within the pool of cellular messages in a
sample. Hybridization analysis can be designed to allow for
concurrent screening of the relative expression of hundreds to
thousands of samples by using, for example, array-based
technologies having high density formats, including filters,
microscope slides, or microchips, or solution-based technologies
that use spectroscopic analysis (e.g., mass spectrometry).
[0058] In other screening methods utilizing cell lysates, the test
sample is assayed for the presence of PTK7 polypeptide. In general,
antibodies that specifically bind PTK7 are added to a sample, and
incubated for a period of time sufficient to allow binding to the
epitope, usually at least about 10 minutes. The antibody can be
detectably labeled for direct detection (e.g., using radioisotopes,
enzymes, fluorescers, chemiluminescers, and the like), or can be
used in conjunction with a second stage antibody or reagent to
detect binding (e.g., biotin with horseradish peroxidase-conjugated
avidin, a secondary antibody conjugated to a fluorescent compound,
e.g. fluorescein, rhodamine, Texas red, etc.) The absence or
presence of antibody binding can be determined by various methods,
including flow cytometry of dissociated cells, microscopy,
radiography, scintillation counting, etc. Any suitable alternative
methods can of qualitative or quantitative detection of levels or
amounts of differentially expressed polypeptide can be used, for
example ELISA, western blot, immunoprecipitation, radioimmunoassay,
etc.
[0059] In some embodiments, the invention provides methods of
determining whether a T-cell in a biological sample is a naive T
cell by detecting the cell surface antigen PTK7. In other
embodiments, the invention provides methods of identifying naive
T-cells by identifying PTK7.sup.+ T cells according to their level
of expression of the PTK7 biomarkers in a biological sample. In
some embodiments, the T cells are CD4.sup.+ T cells. In other
embodiments, the T cells are CD8.sup.+ T cells, e.g. naive T cells
from neonates or infants. Such cells may be further classified as
recent thymic emigrants. Human cells are of particular interest, as
are CD4.sup.+ cells. A biological sample of particular interest is
peripheral blood. In another embodiment, the presence of naive T
cells in a biological sample is determined by quantitating the
level of PTK7 in the sample.
[0060] Where the analysis is based on an intact, e.g. a viable,
cell sample the detection of PTK7 expression is usually based on
immunologic detection, e.g. flow cytometry, immunohistochemistry,
etc. Such analysis may also be used in isolation of viable PTK7+
naive T cells, which cells find use in experimental and therapeutic
methods. The cell sample is typically prepared as a suspension in a
suitable buffer, generally be a balanced salt solution, e.g. normal
saline, PBS, Hank's balanced salt solution, etc., conveniently
supplemented with fetal calf serum or other naturally occurring
factors, in conjunction with an acceptable buffer at low
concentration, generally from 5-25 mM. Convenient buffers include
HEPES, phosphate buffers, lactate buffers, etc.
[0061] Detection or separation of the subject cell population will
typically use immunologic specificity for detection or separation.
Techniques providing accurate separation include fluorescence
activated cell sorters, which can have varying degrees of
sophistication, such as multiple color channels, low angle and
obtuse light scattering detecting channels, impedance channels,
etc. The cells may be selected against dead cells by employing dyes
associated with dead cells (e.g. propidium iodide). Any technique
may be employed which is not unduly detrimental to the viability of
the selected cells. Techniques for affinity separation may include
magnetic separation, using antibody-coated magnetic beads, affinity
chromatography, cytotoxic agents joined to a monoclonal antibody or
used in conjunction with a monoclonal antibody, e.g. complement and
cytotoxins, and "panning" with antibody attached to a solid matrix,
eg. plate, or other convenient technique.
[0062] Of particular interest is the use of antibodies as affinity
reagents. Antibodies may be monoclonal or polyclonal, and may be
produced by transgenic animals, immunized animals, immortalized
human or animal B-cells, cells transfected with DNA vectors
encoding the antibody or T cell receptor, etc. Conveniently, these
antibodies are conjugated with a label for use in detection or
separation. Labels include magnetic beads, which allow for direct
separation, biotin, which can be removed with avidin or
streptavidin bound to a support, fluorochromes, which can be used
with a fluorescence activated cell sorter, or the like, to allow
for ease of separation of the particular cell type. Fluorochromes
that find use include phycobiliproteins, e.g. phycoerythrin and
allophycocyanins, fluorescein and Texas red. Frequently each
antibody is labeled with a different fluorochrome, to permit
independent sorting for each marker.
[0063] The antibodies are added to a suspension of cells, and
incubated for a period of time sufficient to bind the available
cell surface antigens. The incubation will usually be at least
about 5 minutes and usually less than about 30 minutes. It is
desirable to have a sufficient concentration of antibodies in the
reaction mixture, such that the efficiency of the separation is not
limited by lack of antibody. The appropriate concentration is
determined by titration. The medium in which the cells are
separated will be any medium which maintains the viability of the
cells. A preferred medium is phosphate buffered saline containing
from 0.1 to 0.5% BSA. Various media are commercially available and
may be used according to the nature of the cells, including
Dulbecco's Modified Eagle Medium (dMEM), Hank's Basic Salt Solution
(HBSS), Dulbecco's phosphate buffered saline (dPBS), RPMI, Iscove's
medium, PBS with 5 mM EDTA, etc., frequently supplemented with
fetal calf serum, BSA, HSA, etc.
[0064] Methods of sorting cells are well known to persons of
ordinary skill in the art. Cell sorters generally are capable of
separating a complex mixture of cells into fractions of a single
cell type. Typically, the cells to be sorted are introduced as a
thin jet of carrier liquid emanating from a small nozzle orifice.
Shortly after leaving the nozzle, the fluid passes through the
waist of one or more tightly focused laser beams. The scattered and
fluorescence light from these interactions can be collected and
analyzed to determine if there are events (e.g., the presence of a
fluorescence signal indicating that a fluorophore-labeled
monoclonal antibody is bound to the surface of a cell) that prompt
the sorting of the cell by various means. More than one label can
be monitored at a time. FACS (fluorescence activated cell sorters)
can easily analyze cells at speeds greater than 200,000 events per
second. Generally, the physics of the carrier fluid, however, and
the statistics of distributing the cells among the droplets limits
sort rates to about 50,000 cells per second. This combination of
speed and reliable separation allows individual cells to be
isolated for other uses.
[0065] Magnetic cell sorting may be performed using
super-paramagnetic microbeads composed of iron oxide and a
polysaccharide coat. Preferably the microbeads may be approximately
50 nanometers in diameter, and have a volume about one-millionth
that of a typical mammalian cell. The microbeads are preferably
small enough to remain in colloidal suspension, which permits
rapid, efficient binding to cell surface antigens. The microbeads
preferably do not interfere with flow cytometry, are biodegradable,
and have negligible effects on cellular functions. The antibody
coupling to the microbeads may be direct or indirect, via a second
antibody to a ligand such as fluorescein.
[0066] The labeled cells are then separated as to the expression of
cell surface markers as previously described, where an initial
population may be limited to cells that are PTK7+. Optionally the
cell population is then divided into subsets based on expression of
CD3, CD4, and or CD8.
[0067] The separated cells may be collected in any appropriate
medium that maintains the viability of the cells, usually having a
cushion of serum at the bottom of the collection tube. Various
media are commercially available and may be used according to the
nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove's
medium, etc., frequently supplemented with fetal calf serum.
[0068] The invention also provides compositions comprising a
population of cells wherein at least 50% of said cells of said
composition are naive, PTK7.sup.+ T cells. The percentage of naive
T cells in the composition can be ascertained using the methodology
described herein. Preferably, at least 75%, 85%, 90%, 95%, or 98%
of said cells of the composition are naive T cells.
[0069] In some embodiments, the invention further provides an
isolated population of naive T cells that are substantially
CD4.sup.+ and PTK7.sup.+ or PTK7.sup.+ and CD8.sup.+. In some
further embodiments, the isolated population is obtained by
contacting a peripheral blood sample with a labeled antibody
specific for the CD4 biomarker and with a labeled antibody specific
for the PTK7 biomarker to identify naive cells which are CD4.sup.+
and PTK7.sup.+ and isolating the identified cells. In some
embodiments, the CD4 antibody and the PTK7 antibody are each
labeled with a different label. In further such embodiments, the
CD4 antibody label and the PTK7 antibody label are each a
fluorescent label. The CD4.sup.+ and PTK7.sup.+ cells may be
identified and isolated in a fluorescent-activated cell sorter.
[0070] The cells may be administered for therapeutic purposes in
any physiologically acceptable medium, normally intravascularly,
although they may also be introduced into bone or other convenient
site, where the cells may find an appropriate site for activation
and differentiation. Usually, at least 1.times.10.sup.5 cells will
be administered, preferably 1.times.10.sup.6 or more. The cells may
be introduced by injection, catheter, or the like. The cells may be
frozen at liquid nitrogen temperatures and stored for long periods
of time, being capable of use on thawing. If frozen, the cells will
usually be stored in a 10% DMSO, 50% FCS, 40% RPMI 1640 medium.
Once thawed, the cells may be expanded by use of growth factors
and/or stromal cells associated with progenitor cell proliferation
and differentiation.
[0071] The subject cells are useful for in vitro assays and
screening to detect factors that are active on naive T cells. Of
particular interest are screening assays for agents that are active
on human cells. A wide variety of assays may be used for this
purpose, including immunoassays for protein binding; determination
of cell growth, differentiation and functional activity; production
of cytokines, e.g. IFN-.gamma., IL-2; and the like.
[0072] Also provided are reagents and kits thereof for practicing
one or more of the above-described methods. The subject reagents
and kits thereof may vary greatly. In certain embodiments, the kits
include at least a PTK7 antibody. In other embodiments, the kit
includes at least one antibody specific for a T cell marker, e.g.
CD4, CD8, CD3, etc. The antibody may be labeled or the kit may
provide reagents for labeling the antibody. In addition to the
above components, the subject kits will further include
instructions for practicing the subject methods. These instructions
may be present in the subject kits in a variety of forms, one or
more of which may be present in the kit. One form in which these
instructions may be present is as printed information on a suitable
medium or substrate, e.g., a piece or pieces of paper on which the
information is printed, in the packaging of the kit, in a package
insert, etc. Yet another means would be a computer readable medium,
e.g., diskette, CD, etc., on which the information has been
recorded. Yet another means that may be present is a website
address which may be used via the internet to access the
information at a removed site. Any convenient means may be present
in the kits.
[0073] The following examples are included to illustrate the
invention and methods used in practicing the invention, and not to
limit the invention.
EXAMPLES
Protein Tyrosine Kinase 7 is a Novel Marker for CD4+ T-Lineage
Recent Thymic Emigrants Results
[0074] CD4+CD8- thymocytes and cord blood CD4.sup.+ T cells express
high levels of PTK7 mRNA. We reasoned that cord blood CD4.sup.+ T
cells should be highly enriched in RTEs compared to circulating
naive CD4.sup.+ T cells of adults, an idea supported by the sjTREC
content of these two cell populations (FIG. 1A). We hypothesized
that RTEs, including those contained in cord blood CD4.sup.+ T
cells, would have a subset of genes with an expression profile
similar to that of mature CD4.sup.+CD8.sup.- thymocytes and
distinct from the expression profile of adult naive CD4+ T cells.
Two pilot experiments using a small cDNA microarray of
approximately 2500 targets were performed in which the transcript
abundance of adult naive CD4.sup.+ T cells was compared with either
that of post-natal CD4.sup.+CD8.sup.- thymocytes or cord blood
CD4.sup.+ T cells. Using real-time PCR, we confirmed that
transcripts for PTK7 (FIG. 1B), a member of the receptor tyrosine
kinase family and sox-4, a transcription factor involved in
thymocyte development, were expressed at higher levels by cord
blood CD4.sup.+ T cells and CD4.sup.+CD8.sup.-thymocytes compared
to adult naive CD4.sup.+ T cells. We subsequently focused on
evaluating PTK7 as a marker for CD4.sup.+ RTEs, as this protein is
expressed on the cell surface and would potentially be simpler to
evaluate by immunofluorescent antibody staining and flow cytometry
than a nuclear transcription factor.
[0075] PTK7 is expressed on human thymocytes. To confirm the RNA
expression findings at the protein level, we analyzed human
thymocytes for surface expression of PTK7 in conjunction with CD4,
CD8, CD3 and CD1a using a polyclonal PTK7 antibody (Lu et al.
(2004) Nature 430:93-98. The least mature triple-negative
(CD3.sup.-CD4.sup.-CD8.sup.-) subset of thymocytes (TN) showed the
highest expression of PTK7 (FIG. 2 A). In contrast,
CD4.sup.+CD8.sup.+ thymocytes, a more mature subset, had less
expression, and most single-positive CD4.sup.+CD8.sup.- and
CD4.sup.-CD8.sup.+ thymocytes, which include the most mature cells,
had the lowest level of expression. A minority of
CD4.sup.+CD8.sup.- thymocytes had a level of PTK7 staining that was
the same as TNs. These PTK7.sup.high CD4.sup.+CD8.sup.- thymocytes
lacked expression of CD3 (FIG. 2 B), demonstrating that they
constituted an immature single-positive thymocyte population that
in humans is a transition between the triple-negative and CD4+CD8+
stages of thymocyte development. Thus, there was an inverse
relationship between PTK7 surface expression and human thymocyte
maturation.
[0076] We used CD1a staining to further define the relationship
between PTK7 expression level and the maturation of single-positive
thymocytes that had undergone positive selection. CD1a is a
non-classical MHC class I molecule that is progressively lost from
CD3.sup.high single-positive thymocytes as they mature. This is
confirmed in the staining (FIG. 2 C), as the single positive cells
with lowest PTK7 expression also express the lowest levels of CD1a.
Interestingly, there is a population of cells that are
PTK7.sup.-CD1.sup.- that may represent thymic re-entrants (they
also show an intermediate level of CD3 expression FIG. 2 B).
[0077] Murine thymocytes, like those of the human, also showed a
progressive decrease in PTK7 expression with maturation in several
strains (C57BL/6, BALB/c, and FVB/N) (FIG. 9). Previous work has
shown that in most mouse strains, CD3-CD4-CD8+ thymocytes are an
intermediate cell population between the triple-negative and
CD4+CD8+ thymocyte stages. Also, analogous to CD3.sup.+
single-positive human thymocyte maturation, CD4.sup.+CD8.sup.-
single positives that had high levels of PTK7 expression were also
CD24.sup.high, indicating they were less mature and with reduced
function compared to CD24.sup.low cells.
[0078] PTK7 is expressed on a small population of human adult
peripheral naive CD4.sup.+ T cells. Next, we wanted to determine
whether any peripheral T cells maintained a level of expression of
PTK7 after export from the thymus that would mark them as RTEs and
distinguish them from older, more mature naive T cells. As shown in
FIG. 2A, approximately 10-15% of the FSC.sup.loSSC.sup.lo
(lymphocyte) CD4.sup.+CD45RA.sup.hi cells showed expression of PTK7
at low levels. This small percentage of cells was consistently seen
in 4 separate donors of varying ages (ages 24, 25, 30, and 37) and
sexes (2 females, and 2 males) (FIG. 2B). The percentage of cells
expressing PTK7 is consistent with murine models of RTEs that
suggest there is a small amount of thymic output even in two-year
old mice.
[0079] In the human, CD45RA is a tyrosine phosphatase isoform that
marks naive T cells, whereas memory T cells lose CD45RA expression
and begin to express the shorter CD45RO isoform (Clement (1992) J
Clin Immunol 12:1-10). The PTK7.sup.+ shoulder of cells was lost in
the CD45RA.sup.lo population (FIG. 2A), again indicating the
specificity of the staining. In stark contrast, the level of PTK7
expression was uniformly high on nearly all cord blood CD4.sup.+ T
cells (FIG. 2A). Although not all cord blood CD4.sup.+ T cells are
thought to be RTEs, this population is known to be enriched in
RTEs. In addition, cord blood CD8.sup.+ T cells also showed lower,
but still positive levels of PTK7 expression.
[0080] PTK7.sup.+ cells are wholly contained within the "younger"
CD31.sup.+ population, and contain higher TREC levels than either
PTK7-CD31.sup.+ or PTK7CD31.sup.- populations. To confirm that
PTK7.sup.+ cells truly represent RTEs, we co-stained the same sets
of cells for an additional maturation molecule, CD31, which has
been previously characterized with regard to the relative age of
peripheral T cells. The majority of naive CD4+CD45RA.sup.hi T cells
express CD31, which is also an endothelial cell marker (also called
PECAM-1) that contains ITIM domains thought to inhibit TCR
signaling. Sorted populations of CD31.sup.- T cells contain fewer
TRECs than their naive CD31.sup.+ counterparts, and by definition
have undergone more cell divisions (Kimmig et al. (2002) J Exp Med
195:789-794). Therefore, T cells that undergo homeostatic
proliferation (independent of antigen, because they maintain their
naivete) in the periphery lose CD31 expression.
[0081] We hypothesized that the PTK7.sup.+ cells are RTEs, have
undergone the fewest divisions in the periphery (having just
emigrated from the thymus), and would be contained entirely within
the younger CD31.sup.+ population, and not within the CD31.sup.-
"older" population. We stained peripheral T cells from the same
three donors shown in FIG. 2 and gated on lymphocyte
CD4+CD45RA.sup.hi cells, followed by a two-dimensional analysis of
PTK7 and CD31 staining. As shown in FIG. 3, all of the PTK7.sup.+
cells are also CD31.sup.+, and all the CD31.sup.- cells (that have
undergone homeostatic proliferation) are also PTK7.sup.-, as
expected. PTK7.sup.+ and PTK7.sup.- cells express similar levels of
CD5, a marker whose expression level that is unaffected by the
maturation state of the cell, and irrelevant isotype-matched
stained cells are included as a negative control (FIG. 3A).
[0082] To confirm the validity of the staining, we sorted these
three populations of cells (from the same three donors) based on
PTK7 and CD31 co-staining, isolated total DNA from the cells, and
performed an analysis for absolute TREC content, using a
plasmid-based quantitative real-time PCR system on total DNA (Douek
et al. (1998) Nature 396:690-695). TRECs have been used classically
to determine the relative "age" of bulk populations of cells. They
are the signal-joint circular remnants of VDJ recombination
leftover after TCR gene rearrangement in the thymus. Because they
lack an origin of replication, they are not replicated during each
cell division, and as such are diluted in T cells following cell
proliferation (Kong et al. (1998) Immunity 8:97-104).
Unfractionated human thymocytes were used as the positive control
and showed the highest TREC levels (FIG. 3B). The erythroleukemia
cell line K562 was used as the negative control. The
PTK7.sup.-CD31.sup.+ cells (population II) contain higher TREC
levels than the PTK7.sup.-CD31.sup.- cells (population III). RTEs
should contain higher TREC content than older mature naive T cells
since they have undergone fewer cell divisions (because of their
recent thymic origin). Indeed, the level of TRECs in the
PTK7.sup.+CD31.sup.+ cells (population II) is higher than the
PTK7.sup.-CD31.sup.+ cells (population II). The absolute numbers
from these data are consistent with numbers from Kimmig et al.
(2002) J Exp Med 195:789-794. Additional controls for TREC content
from unfractionated CD45RA.sup.+ and CD45RO.sup.+ populations were
included to verify the expected results. Given that the majority of
the CD45RA.sup.+ population is the CD31.sup.+PTK7.sup.- subset, it
is expected that the number of TRECs from these two cell types
would be most similar, and indeed they are (FIG. 3B). The relative
"ages" of human CD4.sup.+ T cells in the periphery can be
delineated as 1. PTK7.sup.+CD31.sup.+ (RTEs), which are the
youngest peripheral CD4.sup.+ T cells recently emigrated from the
thymus, II. PTK7.sup.-CD31.sup.+ (non-RTEs) and III.
PTK7.sup.-CD31.sup.- (non RTEs).
[0083] CD4.sup.+CD25.sup.+FoxP3.sup.+ regulatory T cells do not
express PTK7. To further confirm our finding about the relationship
between PTK7 expression, TREC content, and peripheral age of a cell
based on the number of cell divisions, we wanted to investigate the
expression level of PTK7 on CD4.sup.+CD25.sup.+FoxP3.sup.+
regulatory T cells. It has been shown in previous reports that most
regulatory T cells do not contain TRECs because they undergo
homeostatic proliferation to self antigens in the periphery (Kasow
et al. (2004) J Immunol 172:6123-6128). We therefore hypothesized
that PTK7 would not be expressed on any regulatory T cells in the
periphery. This is, in fact, exactly what we found (FIG. 4). The
permeabilization procedure did not destroy the PTK7 epitope, as
there are still PTK7.sup.+ cells remaining in the stain in the
Foxp3.sup.- populations (FIG. 4). Therefore, the lack of
coexpression of PTK7 and FoxP3 is similar to the lack of
coexpression of PTK7 and CD31, since both populations have
undergone homeostatic proliferation and contain lower TREC
levels.
[0084] Functional characterization of human CD4.sup.+PTK7.sup.+
RTEs. We set out to determine whether adult CD4.sup.+ RTEs differ
in their functional ability as compared to PTK7.sup.- older mature
naive T cells. Previous studies in other species have suggested
that CD4+ and CD8+ RTEs have immaturities in their immune function,
including reduced proliferation and cytokine production in response
to physiological TCR/CD3 stimuli (Boursalian et al. (2004) Nat
Immunol 5:418-425). Human neonatal cord blood CD4.sup.+ T cells,
which have been classified as being enriched in RTEs, have also
been shown to have immaturities in function (Chen et al. (2006)
Biol Blood Marrow Transplant 12:160-171). As proliferation and
cytokine production are the two main components representative of
T-cell activation and differentiation, we sorted both PTK7+ and
PTK7- cells from multiple donors as described previously, then
placed them in culture with .alpha.CD3/28 beads for a 5-day time
course. We measured .sup.3H-Thy incorporation in the final 16 hours
of culture. The PTK7+ cells showed a significant decreased ability
to incorporate radiolabeled nucleotides (FIG. 5). Engagement of the
PTK7 molecule by the antibody itself does not affect the
proliferation of the cells, because cord blood cells proliferate to
the same degree whether they are initially engaged by the antibody
or not (FIG. 5).
[0085] IL-7 is a homeostatic cytokine to which both thymocytes and
cord blood T cells are responsive. Given that PTK7+RTEs are of
recent thymic origin, we hypothesized that they would be more
responsive to IL-7 than their more mature counterparts. This was in
fact what we observed (FIG. 6). Sorted cells from the same donors
were incubated for 3 and 5 days with 10 ng/ml of recombinant human
IL-7, and measured for .sup.3H-thymidine incorporation for the
final 16 hours of culture. The PTK7.sup.+ cells were responsive,
whereas the PTK7.sup.- cells were not. The counts per minute (CPM)
were much lower than those for the TCR stimulated cells. We
performed additional titration of the IL-7 to 50 ng/ml with similar
results. We also carried the cells for 12 days in the presence of
the IL-7, and only got an increase to about 4000 CPM. The
difference in responsiveness to IL-7 was not due to differences in
IL-7R-alpha expression, as both PTK7+RTEs and PTK7- mature
CD4.sup.+ T cells express comparable levels of IL-7R-alpha (FIG.
6).
[0086] To identify additional immaturities in function, we also
investigated the ability of the cells to produce IL-2, TNF-.alpha.,
and IFN-.gamma.. Cells were sorted from the same donors as
described before, and incubated with .alpha.CD3/CD28 beads for 12
hours, then permeabilized and stained for intracellular IL-2 and
TNF-.alpha.. The PTK7.sup.+ cells showed a dramatic reduction in
the percentage of cells positive for IL-2 production (FIG. 7). This
reduction was specific for IL-2, as the percentage of cells
positive for TNF-.alpha. was similar for the two populations. The
reduction in IL-2 production was mirrored in the level of IL-2
protein present in the supernatant, as measured at 12, 24, and 48
hours of culture (FIG. 7).
[0087] Infants are more susceptible to infectious diseases that
require Th1 immunity, and neonatal T cells in the mouse and human
have been shown to have reductions in the ability to produce the
Th1 signature-cytokine, IFN-.gamma.. Because neonatal T cells are
enriched for RTEs, we predicted that adult PTK7+ RTEs would bear an
immaturity in the production of IFN-.gamma.. Because T cells
require a period of time for differentiation into cytokine
expressing cells and also require multiple interactions between T
cells and antigen-presenting cells (APCs) for the induction of
IFN-.gamma., we decided to include autologous MACS-purified
CD14.sup.+ cells at a ratio of 1:10 to the T cells in the
bead-treated cultures as shown in FIG. 8. Following three days of
culture, we collected supernatants and performed IFN-.gamma.
cytokine ELISAs. The PTK7.sup.+ cells showed a drastic reduction in
the ability to produce IFN-.gamma. in response to TCR/CD3 stimuli
in the presence of autologous CD14.sup.+ monocytes.
Materials and Methods.
[0088] Anti-PTK7 antibody. Additional control experiments were
performed to verify the specificity of the anti-serum, including
antibody binding to CHO cells transfected with a GFP-PTK7 fusion
construct, lack of antibody binding to PTK7-/- murine fetal
thymocytes, and additional expression analysis on mouse and human T
cell populations using quantitative real-time PCR, flow cytometry,
and western blotting (FIGS. 10 and 11).
[0089] Microarrays. Microarray experiments were performed as
previously described by Alizadeh et al. (2000) Nature
403:503-511.
[0090] Thymocyte staining. C57BL/6 murine thymus was ground with
frosted slides (and human thymus from pediatric cardiac surgery
patients was ground through a strainer) into a single-cell
suspension, and filtered through a nylon mesh. 1.5.times.10.sup.6
thymocytes were counted and aliquoted into 1.5 ml Eppendorf tubes
with 50 .mu.l of 1.times.PBS pH 7.4 (Gibco), 1% BSA, 0.1% Azide
("PBA"). All staining was performed at room temperature unless
otherwise indicated. Cells were incubated for 20 minutes with
anti-CD16/32 (1:25) (Fc block, Caltag) (or 10% human AB serum for
human cells) for 5 minutes, then with either affinity-purified
anti-mouse PTK7 anti-serum (1:500) or affinity-purified Normal
Rabbit IgG (1:50) (Caltag) as an isotype negative control. Cells
were then washed in 1 ml of PBA, and stained for another 20 minutes
with goat anti-rabbit IgG-FITC (H&L) (1:25) (Caltag). Cells
were washed again, and then stained for 15 minutes at 4 degrees in
Normal Rabbit IgG (1:20). Cells were washed again and then stained
for 10 minutes with anti-CD8, anti-CD4, and anti-CD3. After a final
wash, the cells were fixed with 2% para-formaldehyde in 1.times.PBS
pH 7.4 and analyzed by flow cytometry on a FACScan or FACSCalibur.
CD24-PE from BD Biosciences (clone Ml/69). Human CD1.alpha.-PE from
BD Biosciences.
[0091] Human T-cell staining. PBMC were purified from either human
umbilical cord blood samples (obtained from El Camino Hospital on
scheduled C-sections) or peripheral blood of a 35-year-old male
adult donor by Ficoll Hypaque density gradient purification. Whole
blood was first mixed in a 50:50 ratio with 0.9% NaCl, then
underlaid with Ficoll Hypaque and centrifuged. Interphase harvested
cells were then washed twice with 1.times.HBSS, counted, and 1.5
million cells were stained and analyzed as previously described for
thymocytes, using antibodies to human CD4 and CD45RA. Anti-human
CD31-PE was obtained from BD Biosciences.
[0092] TREC Analysis. Freshly isolated human peripheral blood cells
were prepared, stained, and sorted. Following the sort, cells were
washed once with 1.times.PBS pH 7.4 (Gibco), then total DNA was
prepared using a QIAamp DNA Mini Kit (Qiagen 51304). DNA was then
frozen at -20.degree. C. until use in real time PCR assays. For the
real time PCR assays, a master mix was prepared using Taqman
Universal PCR Master Mix (Roche 4304437), 10 .mu.M forward primer
5'-CCATGCTGACACCTCTGGTT-3', 10 .mu.M reverse primer
5'-TCGTCAGAACGGTGAATGAAG-3', and 10 .mu.M probe FAM-TAMRA
5'-CACGGTGATGCATAGGCACCTGC-3'.
[0093] An equal volume of total DNA from all samples was aliquoted
into the master mix, then plated in triplicate. Plates were
analyzed on the real time machine at 50.degree. C. for 2',
95.degree. C. for 10', then 50 cycles. A standard linear plot was
generated with 10-fold dilutions of a cloned sjTREC starting with
100,000 copies and diluted to 10 copies. Parallel reactions were
used for a cloned portion of TCR.alpha., with 10.0 .mu.M forward
primer 5'-CCTGATCCTCTTGTCCCACAG-3', 10.0 .mu.M reverse primer
5'-GGATTTAGAGTCTCTCAGCTGGTACA-3', and 10.0 .mu.M probe FAM-TAMRA
5'-ATCCAGAACCCTGACCCTGCCG-3'. Values for the linear plot were
either included or excluded to get an R squared value as close to
0.99 as possible, demonstrating an efficiency of amplification of
close to 100%, and thus a Ct difference of 1 representing a 3 fold
difference in copy number. For the calculations, the Ct value for
each sample was interpolated by the machine based on this R squared
value to get an absolute copy number. The calculation for absolute
TREC # was determined with the following formula: # TCR alpha
copies/2=# cells; # sjTREC copies/# cells=# sjTREC copies/cell.
Then, the triplicates were averaged and standard deviation was
performed using Graphpad Prism software. Finally, statistical
comparisons between groups were performed using Prism software with
unpaired student's T test to determine P values.
[0094] The standard TCR.alpha. (T cell receptor alpha constant
region) fragment and the sjTREC fragment were both cloned by PCR
from a preparation of unfractionated human thymocyte DNA into the
TOPO vector. We cloned a 496 bp fragment of TCR alpha constant
region from with 2 primers flanking the DNA sequences used as
primers in the quantitative PCR assay described above. Primers are
5'-ATCACGAGCAGCTGGTTTCT-3', and 5'-CCATTCCTGAAGCAAGGAAA-3'. The 381
bp fragment of sjTREC was cloned with the following 2 primers:
5'-GAAAACAGCCTTTGGGACAC-3', and 5'-GTGACATGGAGGGCTGAACT-3'. Copy
number was calculated using a combination of the OD reading and
length of plasmid product.
[0095] In FIG. 5, the cells were prepared and stained as described
previously. Then cells were fixed, permeabilized, and stained, and
analyzed within 24 hours on a FACScalibur flow cytometer.
[0096] In FIG. 6: Freshly isolated human peripheral blood cells
were prepared, stained, and sorted. Following the sort, cells were
centrifuged and resuspended in RPMI (Gibco), 10% human AB serum,
Pen/Strep/L-Glutamine. Cells were plated into 96 well round bottom
plates in a final volume of 200 .mu.l per well at a final
concentration of 25,000 cells per well. Our own beads or Dynal
beads (anti-CD3/anti-CD28) were added at a final concentration of
0.3 beads or 0.1 beads per cell. IL-7 was used at a final
concentration of 10 ng/ml (from R&D systems). The IL-7 alone
experiments used 50,000 cells per well. .sup.3H-Thymidine was added
on the final 18 hours of culture (1.0 .mu.l per well) prior to
harvesting on Day 2, 3, or 4.
[0097] CD14+ cells were added to ConA treated wells and to
anti-CD3/anti-CD28 bead-treated wells at a concentration of 1:10
that of sorted T cells. Supernatants were collected on Day 3 of
culture, frozen at -80 until, and IL-2, IFN.gamma. were assayed by
ELISA (BD optEIA).
[0098] FIG. 9. Total RNA from thymocytes of a 6-week old male wild
type C57BL/6 mouse was extracted using Tri-reagent (MRC). RNA was
treated with DNase I using an Absolutely RNA Nanoprep kit column
(Stratagene). A cDNA library was then generated with random hexamer
priming and Superscript II RNaseH- (Invitrogen). Mouse PTK7
116-2632 was cloned by PCR using PFU Turbo DNA polymerase
(Stratagene) and the following primers (IDT): (Forward)
5'GGAATTCAGCCACCATGGGAGCCCGCCCGCTG-3', and (Reverse)
5'-TCCCCGCGGACTTCCCGAACATCTCCACC-3'.
[0099] The mouse PTK7 methionine start codon begins with nucleotide
116 and 2632 extends beyond the end of the transmembrane domain
portion of the gene, which ends at nucleotide 2270. The forward
primer above contains an EcoRI site at the 5' end, followed by the
Kozak consensus translation start site ribosome binding site
sequence GCCACC immediately 5' of the 116 PTK7 start codon. The
reverse primer contains a Sac II site immediately 3' of the 2632
region of mPTK7. The restriction sites inserted during the PCR
allowed for directional cloning of this segment of mPTK7 in frame
into the multi-cloning site of pEGFP-N3, originally from Clontech
(Catalog number 6080-1, Genbank accession # U57609). The construct
was sequenced (Bionexus) to confirm 100% nucleotide identity
throughout the designated length of the clone. It was then purified
with the Endofree Plasmid Maxi kit (Qiagen), and digested to verify
the ligation. Then, 10.sup.6 CHO-P cells were transfected by
electroporation with 20 .mu.g pEGFP-N3-PTK7. Briefly, cells were
trypsinized, washed in complete medium, then once in 1.times.PBS,
resuspended in 200 .mu.l DMEM and placed on ice. Time constant was
50.6 and Actual Volts was 0.260. Cells were allowed to recover for
20 minutes on ice prior to plating in complete medium. Cells were
grown for 3 days and analyzed by flow cytometry to verify GFP
fluorescence. Cells were then selected for 2 weeks with 1.0 mg/ml
G418 in medium, changing the media daily. Following two weeks of
selection in G418 to generate the stable transfectant, the cells
were sorted based on GFP fluorescence and prepared and stained as
previously described.
[0100] Timed matings were set up between pairs of PTK7.sup.+/-
mice. To identify heterozygous mice, a small section of tail was
removed with scissors. (All animals were cared for by the rules and
regulations of Stanford Animal standard protocol). Then, tail
sections were incubated with 100 .mu.l of the following solution:
5.0 mM potassium ferricyanide, 5.0 mM potassium ferrocyanide, 2.0
mM magnesium chloride, 2.5 mg/ml beta-galactosidase (X-gal), 0.01%
sodium deoxycholate, 0.02% NP-40, in 1.times.PBS pH 7.4. Tail
sections were incubated for 1 hr in a 37.degree. C. water bath and
genotyped for blue (transgenic) or non-blue (wild-type) appearance.
On Day 18 following impregnation, the mother mouse was sacrificed
by cervical dislocation. Embryos were extracted, and their fetal
thymi were removed under a dissection microscope. PTK7.sup.-/-
embryos were easily identified by the neural tube defect.
Heterozygous and wild type embryos were distinguished by the X-gal
staining method described above using embryo tail sections. Thymi
were then prepared and thymocytes were stained as previously
described.
[0101] FIG. 11. Abbreviations: Mouse: NTC=no-template control,
CD4-CD8- Thy=double negative thymocytes, CD8+CD4+ Thy=double
positive thymocytes, CD4+Thy=CD8-CD4+ single positive thymocytes,
CD4+ LN=CD8-CD4+ peripheral T cells from lymph node (LN), and
EL-4=mouse CD4+ thymoma cell line. Human: CD4+CD1+ Thy=CD8-CD4+CD1+
single positive thymocytes, Cord T cells=unfractionated umbilical
cord blood peripheral T cells, Adult T cells=unfractionated adult
blood peripheral T cells, and Jurkat=CD4+ lymphoma cell line.
[0102] Primers (IDT) for the quantitative PCR reactions are as
follows: Human PTK7 (Exon 4 618-808) (Forward) 620-639:
5'-CCACCTACCAATGGTTCCGA-3'; (Reverse) 650-670:
5'-TGCTCTGACCATCAGAAAGGG-3'. Mouse PTK7 (Exon 20 3141-4054).
(Forward) 3427-2446: 5'-CAGGCATTGCTGAAGACTGG-3'. (Reverse)
3458-3477: 5'-GGTTGTGGCGAAGAGAAACG-3'
[0103] Lysates were prepared from single-cell suspensions of the
indicated cell lines or mouse cell lines. Lysis buffer contained 1%
NP-40, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, and 250 mM NaCl. One
tablet of "Complete Mini" protease inhibitors (Roche) was added per
5 ml lysis buffer. For mouse tissues, 5.0 million cells were used
per 100 .mu.l of lysis buffer, and for cell lines, 1.0 million
cells were used per 100 .mu.l. (Recombinant PTK7 was a gift from X.
Lu). Cells were washed once in 1.times.PBS prior to lysis. After
addition of lysis buffer, cells were vortexed and lysed on ice for
20' prior to centrifuging 20' max speed at 4.degree. C. 1 .mu.l of
lysate in 100 .mu.l of assay buffer in a Bradford assay was
performed to determine the absolute protein concentration. Then 20
.mu.g of total protein per sample was added to sample buffer,
boiled, and loaded onto a 10% SDS-PAGE gel, blotted, blocked, and
probed with the anti-PTK7 antibody. Goat anti-rabbit-HRP (Jackson)
and ECL (KPL) were used for development.
[0104] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes. No reference to a publication herein should be construed
as an admission that such is prior art.
Sequence CWU 1
1
17120DNAArtificial SequencePCR Forward Primer 1ccatgctgac
acctctggtt 20221DNAArtificial SequenceReverse Primer 2tcgtcagaac
ggtgaatgaa g 21323DNAArtificial SequenceProbe FAM-TAMRA 3cacggtgatg
cataggcacc tgc 23421DNAArtificial SequencePCR Forward Primer
4cctgatcctc ttgtcccaca g 21526DNAArtificial SequencePCR Reverse
Primer 5ggatttagag tctctcagct ggtaca 26622DNAArtificial
SequenceProbe FAM-TAMRA 6atccagaacc ctgaccctgc cg
22720DNAArtificial SequencePCR Primer 7atcacgagca gctggtttct
20820DNAArtificial SequencePCR Primer 8ccattcctga agcaaggaaa
20920DNAArtificial SequencePCR Primer 9gaaaacagcc tttgggacac
201020DNAArtificial SequencePCR Primer 10gtgacatgga gggctgaact
201132DNAArtificial SequenceForward PCR Primer 11ggaattcagc
caccatggga gcccgcccgc tg 321229DNAArtificial SequenceReverse PCR
Primer 12tccccgcgga cttcccgaac atctccacc 29136DNAArtificial
SequenceForward PCR Primer Kozak consensus translation start site
ribosome binding site sequence 13gccacc 61420DNAArtificial
SequenceHuman PTK7 Forward Primer 14ccacctacca atggttccga
201521DNAArtificial SequenceHuman PTK7 Reverse Primer 15tgctctgacc
atcagaaagg g 211620DNAArtificial SequenceMouse PTK7 Forward Primer
16caggcattgc tgaagactgg 201720DNAArtificial SequenceMouse PTK7
Reverse Primer 17ggttgtggcg aagagaaacg 20
* * * * *